Cytochrome c oxidase is an oligomeric protein complex isolated from the inner mitochondrial membrane of eucaryotic organisms. It is responsible for the terminal step in electron transport from cytochrome c to molecular oxygen. The energy derived from this electron flow is coupled to the conservation of energy in the form of adenosine triphosphate through oxidative phosphorylation. As such, cytochrome c oxidase is an enzyme of immense importance and plays a key role in cell respiration. This project will provide structural information about the three-dimensional arrangement of cytochrome c oxidase through the measurement of distances between protein sites, lipid sites and substrate sites in wild-type and functional structural mutants of cytochrome c oxidase isolated from yeast. The immediate objectives of the proposal are: (1) to chemically modify specific cytochrome c oxidase sites with fluorescent and nonfluorescent chromophores; (2) to reconstitute these labeled cytochrome c oxidase molecules into defined phospholipid vesicles; (3) using the technique of fluorescence resonance energy transfer, measure the spacial relationships between these labeled sites, intrinsic chromophores and covalently attached substrates in isolated and vesicular cytochrome c oxidase; (4) to determine the changes in these spacial relationships in functional mutants of cytochrome c oxidase which contain a single altered polypeptide chain; and (5) correlate these special relationships using reversible crosslinking reagents. The end result of this investigation will be a detailed structural map of the three-dimensional arrangement of the subunits of cytochrome c oxidase with relation to the subunits themselves, to intrinsic heme groups, substrate and to lipid environment. In turn, the study will contribute information about the mechanism of cytochrome c oxidase electron flow as well as mitochondrial membrane biogenesis.